C-terminal Tyrosine Phosphorylation Research Articles

Suppression of human STAT2 by ZIKV NS5

Abstract The infection of ZIKA virus (ZIKV), a mosquito-transmitted flavivirus, has been shown to cause congenital disease in the context of infection during pregnancy in its 2015 outbreak. To establish the infection, ZIKV must suppress host interferon (IFN) response. Previous studies have shown that ZIKV utilizes its non-structural NS5 protein to trigger the human STAT2, a critical mediator to boost host interferon (IFN) response, for the proteasome-dependent degradation. However, the underlying mechanisms of viral NS5 and hSTAT2 remain elusive largely due to the lack of atom level conformational information. Here, we report the structure of hSTAT2 in complex with ZIKV NS5. hSTAT2, with the SH2 domain occluded by the C-terminal tyrosine-phosphorylation (pY)-tail segment, inserts the coiled-coil domain into the cleft between the methyltransferase and RdRP domains of NS5 in a manner that would occlude the association of hSTAT2 with cellular regulatory protein IRF9. Disruption of the ZIKV NS5 – hSTAT2 association in cells compromises the NS5-mediated hSTAT2 degradation and IFN responses. This study provides a basis for understanding the functional antagonism between viral antagonists and STAT2.

Shp2-Mitogen-Activated Protein Kinase Signaling Drives Proliferation during Zebrafish Embryo Caudal Fin Fold Regeneration.

ABSTRACTRegeneration of the zebrafish caudal fin following amputation occurs through wound healing, followed by formation of a blastema, which produces cells to replace the lost tissue in the final phase of regenerative outgrowth. We show that ptpn11a−/− ptpn11b−/− zebrafish embryos, lacking functional Shp2, fail to regenerate their caudal fin folds. Rescue experiments indicated that Shp2a has a functional signaling role, requiring its catalytic activity and SH2 domains but not the two C-terminal tyrosine phosphorylation sites. Surprisingly, expression of Shp2a variants with increased and reduced catalytic activity, respectively, rescued caudal fin fold regeneration to similar extents. Expression of mmp9 and junbb, indicative of formation of the wound epidermis and distal blastema, respectively, suggested that these processes occurred in ptpn11a−/− ptpn11b−/− zebrafish embryos. However, cell proliferation and MAPK phosphorylation were reduced. Pharmacological inhibition of MEK1 in wild-type zebrafish embryos phenocopied loss of Shp2. Our results suggest an essential role for Shp2a–mitogen-activated protein kinase (MAPK) signaling in promoting cell proliferation during zebrafish embryo caudal fin fold regeneration.

Sulfiredoxin Promotes Colorectal Cancer Cell Invasion and Metastasis through a Novel Mechanism of Enhancing EGFR Signaling.

Sulfiredoxin (SRXN1/Srx) is a multifunction enzyme with a primary antioxidant role of reducing the overoxidized inactive form of peroxiredoxins (Prxs). The function and mechanisms of Srx in cancer development are not well understood. Here, Srx is preferentially expressed in human colorectal cancer cells but not in normal colon epithelial cells. Loss-of-function studies demonstrate that knockdown of Srx in poorly differentiated colorectal cancer cells not only leads to the inhibition of colony formation and cell invasion in vitro, but also reduces tumor xenograft growth and represses metastasis to distal organs in a mouse orthotopic implantation model. Notably, exactly opposite effects were observed in gain-of-function experiments when Srx was ectopically expressed in well-differentiated colorectal cancer cells. Mechanistically, expression of Srx enhances the activation of MAPK signaling through increasing the C-terminal tyrosine phosphorylation levels of EGFR. This function of Srx is mediated through its inhibition of EGFR acetylation at K1037, a novel posttranslational modification of EGFR in human colorectal cancer cells identified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI/MS-MS) proteomic analysis. Furthermore, abolishment of K1037 acetylation in human colorectal cancer cells by site-specific mutagenesis leads to sustained activation of EGFR-MAPK signaling. Combined, these data reveal that Srx promotes colorectal cancer cell invasion and metastasis through a novel mechanism of enhancing EGFR signaling. Sulfiredoxin is a critical oncogenic protein that can be used as a molecular target to develop therapeutics for patients with metastatic colorectal cancer.

Phosphorylation of C-terminal tyrosine residue 526 in FUS impairs its nuclear import.

Aberrant cytoplasmic aggregation of FUS, which is caused by mutations primarily in the C-terminal nuclear localisation signal, is associated with 3% of cases of familial amyotrophic lateral sclerosis (ALS). FUS aggregates are also pathognomonic for 10% of all frontotemporal lobar degeneration (FTLD) cases; however, these cases are not associated with mutations in the gene encoding FUS. This suggests that there are differences in the mechanisms that drive inclusion formation of FUS in ALS and FTLD. Here, we show that the C-terminal tyrosine residue at position 526 of FUS is crucial for normal nuclear import. This tyrosine is subjected to phosphorylation, which reduces interaction with transportin 1 and might consequentially affect the transport of FUS into the nucleus. Furthermore, we show that this phosphorylation can occur through the activity of the Src family of kinases. Our study implicates phosphorylation as an additional mechanism by which nuclear transport of FUS might be regulated and potentially perturbed in ALS and FTLD.

Helicobacter pylori infection activates Src homology-2 domain-containing phosphatase 2 to suppress IFN-γ signaling.

Helicobacter pylori infection not only induces gastric inflammation but also increases the risk of gastric tumorigenesis. IFN-γ has antimicrobial effects; however, H. pylori infection elevates IFN-γ-mediated gastric inflammation and may suppress IFN-γ signaling as a strategy to avoid immune destruction through an as-yet-unknown mechanism. This study was aimed at investigating the mechanism of H. pylori-induced IFN-γ resistance. Postinfection of viable H. pylori decreased IFN-γ-activated signal transducers and activators of transcription 1 and IFN regulatory factor 1 not only in human gastric epithelial MKN45 and AZ-521 but also in human monocytic U937 cells. H. pylori caused an increase in the C-terminal tyrosine phosphorylation of Src homology-2 domain-containing phosphatase (SHP) 2. Pharmacologically and genetically inhibiting SHP2 reversed H. pylori-induced IFN-γ resistance. In contrast to a clinically isolated H. pylori strain HP238, the cytotoxin-associated gene A (CagA) isogenic mutant strain HP238(CagAm) failed to induce IFN-γ resistance, indicating that CagA regulates this effect. Notably, HP238 and HP238(CagAm) differently caused SHP2 phosphorylation; however, imaging and biochemical analyses demonstrated CagA-mediated membrane-associated binding with phosphorylated SHP2. CagA-independent generation of reactive oxygen species (ROS) contributed to H. pylori-induced SHP2 phosphorylation; however, ROS/SHP2 mediated IFN-γ resistance in a CagA-regulated manner. This finding not only provides an alternative mechanism for how CagA and ROS coregulate SHP2 activation but may also explain their roles in H. pylori-induced IFN-γ resistance.

Src Kinase Can Activate RUNX1 Activity Via Phosphorylation Of C-Terminal Tyrosines and Activated RUNX1 Stimulates Granulopoiesis

We demonstrated that G-CSF signaling in lineage-negative marrow myeloid progenitors induces C-terminal SHP2 tyrosine phosphorylation more potently than does M-CSF signaling and that SHP2 knockdown in marrow or in the 32Dcl3 cell line reduces Cebpa gene transcription and impairs granulopoiesis.1,2 We also found that Runx1 directly activates Cebpa transcription via promoter elements and via a +37 kb enhancer and that Runx1 gene deletion reduces Cebpa mRNA and impairs granulopoiesis.3 Runx1 is phosphorylated by Src kinase on five tyrosines and dephosphorylated by SHP2, and RUNX1(5F) enhances megakaryopoiesis and rescues CD8 T cell development, whereas RUNX1(5D) is ineffective.4 Mutation to F prevents while change to D or E mimics phosphorylation. These findings suggested that in myeloid cells there exists a G-CSF to SHP2 to RUNX1 to Cebpa activation pathway directing granulopoiesis. We compared wild-type (WT) murine RUNX1b and a series of variants, Y260/375/378/379/386F (5F), Y260F (1F), Y375/378/379/386F (4F), 5D, 1D, and 4D for activation in 293T cells of a reporter with four RUNX1-binding sites linked to the TK promoter and the luciferase cDNA. Expression was compared by Western blotting. WT stimulated the reporter 5-fold on average, relative to empty CMV vector, 5D activated the reporter 15-fold, while 5F was nearly inactive. 4F was also nearly inactive, while 4D had WT activity, as did the 1F and 1D variants. Both 4F and 5F were expressed at or above WT levels; interestingly, 5D, but not 4D or 1D, was expressed at much higher levels than the other variants. A similar pattern was obtained with the MCSFR-Luc reporter. To further query the activity of the four C-terminal tyrosines, we evaluated the activity of Y375/378F (2F*), Y379/386F (2F), 2E, Y375F, Y378F, Y379F, and Y386F. The single residue mutations retained WT activity, whereas both the 2F* and 2F variants demonstrated significant reduction despite expression at or above WT levels. The 2E variant expressed at levels below WT but had increased activity. We then transfected the RUNX1 reporter with activated Src, Runx1, or both – in several experiments Src stimulated the reporter approximately 3-fold, on average, RUNX1 4-fold, and the combination 35-fold. In the same experiments, Src + 5F led to only 6.6-fold activation, and synergy was also lost if the reporter lacked the RUNX1-binding sites. These findings, together with the observation that SHP2 directly activates Src family kinases downstream of G-CSF receptor signaling,5 raises the possibility that in myeloid cells there actually is a G-CSF to SHP2 to Src to Runx1 to Cebpa activation pathway. Consistent with this idea, we find that the 5D variant has reduced interaction with co-expressed HDAC3 relative to WT, as assessed by co-IP, whereas the 5F variant has increased interaction. We previously found that RUNX1-ER stimulates endogenous Cebpa transcription when activated in 32Dcl3 cells. As the ER segment has trans-activating activity, to evaluate RUNX1 variants we utilizing the zinc-inducible MT promoter. Preliminary analysis indicates that both WT RUNX1b and its 5D variant can induce C/EBPa protein and RNA in this context. We previously found that exogenous RUNX1-ER can rescue granulopoiesis when transduced into marrow from Runx1(flox/flox);Mx1-Cre mice subjected 4 weeks earlier to pIpC injections. We now have constructed MIGC retroviral vectors expressing RUNX1, 5F, or 5D and GFP-Cre. These, or the empty MIG or MIGC vectors, were packaged and transduced into marrow isolated from RUNX1(flox/flox) mice. Three days later, lineage-depleted, GFP+ cells were cultured in methylcellulose with IL-3, IL-6, and SCF. Compared with MIG, MIGC reduced the percent CFU-G amongst CFU-G+CFU-M, from 55+/-1% to 10+/-2%; WT RUNX1 or 5D partially rescued granulopoiesis, to 31+/- 8% or 23+/-9% respectively, whereas 5F transduction led to only 4+/-1% CFU-G. In summary, in 293T cells and potentially in myeloid cells Src kinase activates Runx1 and this may favor granulopoiesis.1. Jack et al. Blood 114:2172, 2009. 2. Zhang and Friedman. Blood 118:2266, 2011. 3. Guo et al. Blood 119:4408, 2012. 4. Huang et al. Genes Dev. 26:1587, 2012. 5. Futami et al. Blood 118:1077, 2011. Disclosures:Cantor:AMGEN: Membership on an entity's Board of Directors or advisory committees.

Constitutive activation of breast tumor kinase accelerates cell migration and tumor growth in vivo

Breast tumor kinase (BRK) is a non-receptor tyrosine kinase overexpressed in most human breast tumors, including lymph node metastases, but undetected in normal mammary tissue or in fibroadenomas. The activity of BRK-like Src family tyrosine kinase, is regulated negatively by phosphorylation of C-terminal tyrosine 447. Although the kinase that regulates BRK activation has not been identified, we and others have previously shown that BRK-Y447F is a constitutively active variant. Because BRK-Y447F significantly enhances the catalytic activity of the enzyme, we investigated the role of the constitutively active BRK variant in tumor formation and metastasis. Using stable breast cancer cell MDA-MB-231 we observed significantly enhanced rates of cell proliferation, migration and tumor formation in BRK-Y447F stable cells compared with wild-type stable cell lines. Our results indicate full activation of BRK is an essential component in the tumorigenic role of BRK.

Regulation of the Rho Family Small GTPase Wrch-1/RhoU by C-Terminal Tyrosine Phosphorylation Requires Src

Wrch-1 is an atypical Rho family small GTPase with roles in migration, epithelial cell morphogenesis, osteoclastogenesis, and oncogenic transformation. Here, we observed rapid relocalization of Wrch-1 from the plasma membrane upon serum stimulation. Studies revealed a requirement for serum-stimulated tyrosine phosphorylation of Wrch-1 at residue Y254 within its C-terminal membrane targeting domain, mediated by the nonreceptor tyrosine kinase Src. Genetic or pharmacological loss of Src kinase activity blocked both phosphorylation and relocalization of Wrch-1. Functionally, Y254 was required for proper Wrch-1 modulation of cystogenesis in three-dimensional culture, and the phospho-deficient mutant, Y254F, was enhanced in Wrch-1-mediated anchorage-independent growth. Mechanistically, C-terminal tyrosine phosphorylation and subsequent relocalization of Wrch-1 downregulated its ability to interact with and activate its effectors by decreasing active Wrch-1-GTP, perhaps by altering proximity to a GEF or GAP. Phospho-deficient Wrch-1(Y254F) remained at the plasma membrane and GTP bound and continued to recruit and activate its effector PAK, even upon serum stimulation. In contrast, a phospho-mimetic mutant, Y254E, was constitutively endosomally localized and GDP bound and failed to recruit PAK unless mutated to be constitutively active/GAP insensitive. C-terminal tyrosine phosphorylation thus represents a new paradigm in posttranslational control of small GTPase localization, activation, and biological function.

Abstract 4002: C-terminal phosphorylation of human GSTP1 by EGFR and site-directed mutation allows JNK reactivation in glioma cells

Abstract In malignant gliomas, the c-jun N-terminal kinase (JNK) pathway regulates tumor growth and resistance to apoptosis, and correlates with histologic grade and epidermal growth factor receptor (EGFR) tyrosine kinase expression. We recently reported that glutathione S-transferase P1 (GSTP1), a major metabolizing and stress response signaling protein that is frequently overexpressed in brain tumors, is a downstream target of EGFR and undergoes EGFR-dependent tyrosine phosphorylation, in vitro and in vivo, resulting in an enhancement of its enzymatic function and increased drug resistance. Although the dimeric GSTP1 exists in a reversible equilibrium with its monomeric form, the monomeric GSTP1 binds to JNK and acts as an endogeneous JNK inhibitor. In our previous study, the EGFR-phosphorylation of GSTP1 shifted the GSTP1 dimer-monomer equilibrium toward the monomeric state and the c-Jun phosphorylation by JNK was enhanced by phosphorylated-GSTP1 and suppressed by unphosphorylated-GSTP1. EGFR activation resulted in dissociation of GSTP1 from the GSTP1-JNK1 complex in a cell-free system and in glioma cells. The C-terminal domain of GSTP1 is reported to be involved in the interaction with JNK. Accordingly, we synthesized the C-terminal domain peptides of human GSTP1 containing the phospho-acceptor Tyr198, unique to hominidae among species, by EGFR (Y198WT) and its phenylalanine-replaced mutant peptide (Y198F). Both the C-terminal domain peptides significantly suppressed the c-Jun phosphorylation by JNK1 in a cell-free system. The binding assay using the synthetic peptides showed that the C-terminal domain peptides of human GSTP1 bound to JNK1 with a lower affinity when Tyr 198 was phosphorylated by EGFR than when it was unphosphorylated. Mutagenesis in human glioma cells revealed that the EGF-induced c-Jun phosphorylation by JNK decreased in the GSTP1-Y198F mutant, whereas increased in the phospho-mimic GSTP1-Y198D mutant, compared with vector control. Together these results indicate that in malignant glioma cells the C-terminal tyrosine phosphorylation of human GSTP1 by EGFR leads to dissociation of GSTP1 from the GSTP1-JNK1 complex. Thus, the GSTP1-JNK1 direct interaction rather than JNK upstream signaling elicits reactivation of JNK. This regulation of JNK signaling function by the EGFR-GSTP1 crosstalk, defines a novel signaling network with a potential to regulate the biology and therapeutic outcome in malignant gliomas. Supported by NIH grants RO1 CA127872, RO1 CA 112519, P50CA108786 and P30-CA14236. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4002.

Regulation of the Rho Family Small GTPase Wrch-1/RhoU by C-Terminal Tyrosine Phosphorylation Requires Src

Regulation of the Rho Family Small GTPase Wrch-1/RhoU by C-Terminal Tyrosine Phosphorylation Requires Src

The lectin Jacalin induces human B‐lymphocyte apoptosis through glycosylation‐dependent interaction with CD45

It has been well established that CD45 is a key receptor-type protein tyrosine phosphatase (PTPase) regulating Src-family protein tyrosine kinase (Src-PTK) in T and B lymphocytes. However, precisely how CD45 exerts its effect in these lymphocytes remains controversial. We recently reported that Jacalin, an alpha-O-glycoside of the disaccharide Thomsen-Friedenreich antigen-specific lectin from jackfruit seeds, caused marked T-cell activation in response to T-cell receptor ligation and CD28 costimulation by binding to CD45. On extending the reported research, we found that CD45 and isoforms are major Jacalin receptors on B lymphocytes, and that the glycosylation of CD45 is involved in the interaction of Jacalin with the PTPase. In contrast to Jacalin-stimulated T-cell activation, we found that Jacalin induced human B-lymphocyte apoptosis, resulting in calcium mobilization and calpain activation, suggesting that the calcium-calpain pathway may mediate the Jacalin-induced apoptosis. Importantly, the apoptosis was effectively blocked by a specific CD45 PTPase inhibitor, indicating that Jacalin induces human B-lymphocyte apoptosis through CD45 triggering. Furthermore, we found that Jacalin significantly increased the C-terminal inhibitory tyrosine (Tyr507) phosphorylation of Src-PTK Lyn, one of the major substrates of CD45 PTPase, and this effect was also observed on incubation of B lymphocytes with the specific CD45 PTPase inhibitor, suggesting that Jacalin stimulation results in increasing C-terminal tyrosine phosphorylation of the kinase through inhibition of CD45 tyrosine phosphatase activity in human B lymphocytes. Therefore, the down-modulation of Lyn kinase may play a role in the regulation of B-lymphocyte viability.

Activation of c-Src and Fyn Kinases by Protein-tyrosine Phosphatase RPTPα Is Substrate-specific and Compatible with Lipid Raft Localization

Src family tyrosine kinases (SFKs) function in multiple signaling pathways, raising the question of how appropriate regulation and substrate choice are achieved. SFK activity is modulated by several protein-tyrosine phosphatases, among which RPTPalpha and SHP2 are the best established. We studied how RPTPalpha affects substrate specificity and regulation of c-Src and Fyn in response to epidermal growth factor and platelet-derived growth factor. We find that RPTPalpha, in a growth factor-specific manner, directs the specificity of these kinases toward a specific subset of SFK substrates, particularly the focal adhesion protein Paxillin and the lipid raft scaffolding protein Cbp/PAG. A significant fraction of RPTPalpha is present in lipid rafts, where its targets Fyn and Cbp/PAG reside, and growth factor-mediated SFK activation within this compartment is strictly dependent on RPTPalpha. Forced concentration of RPTPalpha into lipid rafts is compatible with activation of Fyn. Finally, RPTPalpha-induced phosphorylation of Paxillin and Cbp/PAG induces recruitment of the SFK inhibitory kinase Csk, indicative of negative feedback loops limiting SFK activation by RPTPalpha. Our findings indicate that individual SFK-controlling PTPs play important and specific roles in dictating SFK substrate specificity and regulatory mechanism.

Focal Adhesion Kinase Is Negatively Regulated by Phosphorylation at Tyrosine 407

Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs via tyrosine phosphorylation at specific residues. Although several tyrosine phosphorylation events have been linked to FAK activation and downstream signal transduction, the function of FAK phosphorylation at Tyr(407) was previously unknown. Here, we show for the first time that phosphorylation of FAK Tyr(407) increases during serum starvation, contact inhibition, and cell cycle arrest, all conditions under which activating FAK Tyr(397) phosphorylation decreases. Transfection of NIH3T3 cells with a phosphorylation-mimicking FAK 407E mutant decreased autophosphorylation at Tyr(397) and inhibited both FAK kinase activity in vitro and FAK-mediated functions such as cell adhesion, spreading, proliferation, and migration. The opposite effects were observed in cells transfected with nonphosphorylatable mutant FAK 407F. Taken together, these data suggest the novel concept that FAK Tyr(407) phosphorylation negatively regulates the enzymatic and biological activities of FAK.

C-Cbl-facilitated cytoskeletal effects in v-Abl-transformed fibroblasts are regulated by membrane association of c-Cbl

The multi-functional protein c-Cbl is an important modulator of actin cytoskeletal dynamics in diverse biological systems. We had previously reported that c-Cbl facilitates cell spreading and adhesion and suppresses anchorage-independent growth of v-Abl-transformed fibroblasts. To assess the importance of membrane localization of c-Cbl for the observed effects of c-Cbl in v-Abl-3T3 cells, we first mapped the membrane interactive domain(s) of c-Cbl. Our studies indicate that localization of c-Cbl to the membrane is likely to be mediated by the tyrosine kinase binding (TKB) domain and the proline-rich region of c-Cbl, whereas C-terminal tyrosine phosphorylation does not play a role. The association of v-Cbl, which encompasses the TKB domain, with the membrane was unusual as it was not entirely dependent on SH2-phosphotyrosine interactions. Our studies further demonstrate that Src-like adaptor protein (SLAP), which binds to v-Cbl in a tyrosine phosphorylation-independent manner, facilitates membrane association of Cbl. The interaction between c-Cbl and SLAP in v-Abl-3T3 cells positively influenced c-Cbl-mediated spreading and adhesion of these cells. SLAP appears to exert its effects not simply by increasing the amount of c-Cbl in the membrane but by facilitating binding of p85-phosphatidylinositol-3-kinase (PI3K) with membrane-associated c-Cbl.

Reconstitution of Regulated Phosphorylation of FcϵRI by a Lipid Raft-excluded Protein-tyrosine Phosphatase

To examine the exquisite regulation of IgE-FcepsilonRI tyrosine phosphorylation by Lyn kinase that is stimulated by antigen-mediated cross-linking, we utilized co-expression of FcepsilonRI and Lyn in Chinese hamster ovary cells, which results in high basal levels of Lyn kinase activity and spontaneous phosphorylation of FcepsilonRI. We found that co-expression of a lipid raft-excluded transmembrane tyrosine phosphatase, PTPalpha, suppresses Lyn kinase activity and markedly reduces the level of spontaneous phosphorylation of FcepsilonRI, while facilitating its antigen-stimulated phosphorylation. Other tyrosine phosphatases, including SHP-1, CD45, and a lipid raft-preferring chimeric version of PTPalpha fail to reconstitute antigen-dependent FcepsilonRI phosphorylation. We concluded that both substrate specificity and submembrane location are critical to phosphatase-mediated regulation of Lyn kinase activity that supports activation of FcepsilonRI.

PVR plays a critical role via JNK activation in thorax closure during Drosophila metamorphosis.

PVR, the Drosophila homolog of the PDGF/VEGF receptor, has been implicated in border cell migration during oogenesis and hemocyte migration during embryogenesis. It was earlier shown that Mbc, a CDM family protein, and its effector, Rac, transduced the guidance signal from PVR during border cell migration. Here we demonstrate that PVR is also required for the morphogenetic process, thorax closure, during metamorphosis. The results of genetic and biochemical experiments indicate that PVR activates the JNK pathway. We present evidence showing Crk (an adaptor molecule), Mbc, ELMO (a homolog of Caenorhabditis elegans CED-12 and mammalian ELMO), and Rac to be mediators of JNK activation by PVR. In addition, we suppose that not only Rac but also Cdc42 is activated and involved in JNK activation downstream of PVR.

Cold-inducible expression of the cell division cycle gene CDC48 and its promotion of cell proliferation during cold acclimation in zebrafish cells

A member of the ATPases associated with diverse cellular activities (AAA) family, the cell division cycle gene CDC48/VCP (valosin-containing protein)/p97, was cloned from zebrafish and found to be a major cold-inducible protein in fish cells. CDC48 mRNA levels increased significantly after reducing the temperature from 30 to 15°C for 25 days. CDC48 protein levels also increased 2.5-fold after 30 days at cold temperatures. When fish cells overexpressing CDC48 were exposed to a temperature of 15°C, cell proliferation was markedly enhanced in comparison with control cells. By contrast, expression of a mutant molecule with a tyrosine-805 to alanine substitution at the C-terminal phosphorylation site inhibited cell proliferation and induced apoptosis at low temperatures. Therefore, CDC48 may promote cell cycling and cell proliferation via C-terminal tyrosine phosphorylation during cold acclimation in fish cells.

The Role of C-terminal Tyrosine Phosphorylation in the Regulation of SHP-1 Explored via Expressed Protein Ligation

The protein-tyrosine phosphatase SHP-1 plays a variety of roles in the "negative" regulation of cell signaling. The molecular basis for the regulation of SHP-1 is incompletely understood. Whereas SHP-1 has previously been shown to be phosphorylated on two tail tyrosine residues (Tyr(536) and Tyr(564)) by several protein-tyrosine kinases, the effects of these phosphorylation events have been difficult to address because of the intrinsic instability of the linkages within a protein-tyrosine phosphatase. Using expressed protein ligation, we have generated semisynthetic SHP-1 proteins containing phosphotyrosine mimetics at the Tyr(536) and Tyr(564) sites. Two phosphonate analogues were installed, phosphonomethylenephenylalanine (Pmp) and difluorophosphonomethylenephenylalanine (F(2)Pmp). Incorporation of Pmp at the 536 site led to 4-fold stimulation of the SHP-1 tyrosine phosphatase activity whereas incorporation at the 564 site led to no effect. Incorporation of F(2)Pmp at the 536 site led to 8-fold stimulation of the SHP-1 tyrosine phosphatase activity and 1.6-fold at the 564 site. A combination of size exclusion chromatography, phosphotyrosine peptide stimulation studies, and site-directed mutagenesis led to the structural model in which tyrosine phosphorylation at the 536 site engages the N-Src homology 2 domain in an intramolecular fashion relieving basal inhibition. In contrast, tyrosine phosphorylation at the 564 site has the potential to engage the C-Src homology 2 domain intramolecularly, which can modestly and indirectly influence catalytic activity. The finding that phosphonate modification at each of the 536 and 564 sites can promote interaction with the Grb2 adaptor protein indicates that the intramolecular interactions fostered by post-translational modifications of tyrosine are not energetically strong and susceptible to intermolecular competition.

造血幹細胞数および造血能を制御する抑制性アダプター蛋白質Ｌｎｋ

Hematopoietic stem cells (HSCs) give rise to variety of hematopoietic cells, and are responsible for blood production throughout adult life. Recent studies have demonstrated the potential of HSCs to produce various nonhematopoietic cells. Amplification of HSCs represents a potentially powerful approach not only to the treatment of various blood disorders but also to applying gene therapy or to regenerative medicine. Lnk is an adaptor protein consisting of PH, SH2 domains, and a C-terminal tyrosine phosphorylation site. Marked expansion of Blineage cells occurs in lnk-/- mice, indicating Lnk regulates B cell production by negatively controlling growth signals through c-Kit in B cell precursors. Lnk is also expressed in hematopoietic progenitors, and the number and the hematopoietic ability of HSCs are significantly increased in the absence of Lnk. While augmented signaling through c-Kit partly contributed to the increased progenitor cells in lnk-/- mice, it is likely that there exist Lnkdependent but c-Kit-independent signaling pathways whose absence leads to augmented ability of lnk-/- HSCs. These indicate that Lnk plays critical roles in the expansion and function of HSCs and provide useful clues for the amplification of HSCs and progenitor cells.

Membrane Localization of Src Homology 2-Containing Inositol 5′-Phosphatase 2 via Shc Association Is Required for the Negative Regulation of Insulin Signaling in Rat1 Fibroblasts Overexpressing Insulin Receptors

Lipid phosphatase SHIP2 [Src homology 2 (SH2)-containing inositol 5'-phosphatase 2] has been shown to be a physiologically critical negative regulator of insulin signaling. We investigated the molecular mechanism by which SHIP2 negatively regulates insulin-induced phosphorylation of Akt, a key downstream molecule of phosphatidylinositol 3-kinase important for the biological action of insulin. Overexpression of wild-type SHIP2 (WT-SHIP2) inhibited insulin-induced phosphorylation of Akt at both Thr(308) and Ser(473) in Rat1 fibroblasts expressing insulin receptors. The degree of inhibition was less in the cells expressing either a mutant SHIP2 with R47Q change (R/Q-SHIP2) in the SH2 domain, or a mutant SHIP2 with Y987F change (Y/F-SHIP2) in the C-terminal tyrosine phosphorylation site. However, on addition of a myristoylation signal, WT-SHIP2, R/Q-SHIP2, and Y/F-SHIP2 all efficiently inhibited insulin-induced Akt phosphorylation at both residues, whereas a 5'-phosphatase-defective mutant SHIP2 (deltaIP-SHIP2) with the myristoylation signal did not. Interestingly, the degree of inhibition of Akt phosphorylation by R/Q-SHIP2 and Y/F-SHIP2 is well correlated with the extent of their association with Shc. In addition, overexpression of WT-Shc increased the insulin-induced association of SHIP2 with Shc, whereas a decrease in the amount of Shc on expression of antisense Shc mRNA led to a reduction in the SHIP2-Shc association. Furthermore, the inhibitory effect on insulin-induced Akt phosphorylation by WT-SHIP2 was decreased in antisense-Shc cells. These results indicate that the membrane localization of SHIP2 with its 5'-phosphatase activity is required for negative regulation of insulin-induced Akt phosphorylation and that the localization is regulated, at least in part, by the association of SHIP2 with Shc in Rat1 fibroblasts.